THG was initially discovered as a urinary mucoprotein (Morner Kah, 1895, Skand. Arch. Physiol. 6:332) and was thereafter reported to be a factor acting to inhibit in urine viral hemagglutination (Tamm, I. and Horsfall, E. L., 1950, Proc. Soc. Exp. Biol. Med., 74: 108). Referring specifically to the purification process for THG, a pool of urine collected is stored at 4.degree. C. overnight, and the resultant supernatant as separated out, after a portion of the same is discarded, is admixed with sodium chloride to a concentration of 0.58 M, followed by stirring and centrifugation; the resultant precipitate is collected and dissolved in water, followed by centrifugation, and the supernatant is collected and freeze-dried to thereby give a lyophilisate, which is regarded as THG. Subsequently, THG has been purified by means of a variety of the modified processes devised by a large number of researchers, whereby purification is fundamentally effected along the line of the above-described procedure (Moonen, P. et al., FEBS Lett., 226:314. Toma, G. et al., 1994, Biochem. Biophys. Res. Commun., 200: 275).
On the other hand, uromodulin, a glycoprotein showing the same amino acid sequence as THG, was purified from urine of pregnant women (Muchmore, A. V. and Decker, J. M., 1985, Science, 229:479). Pregnancy urine was subjected to a lectin column (Con A-Sepharose column), and after elution with .alpha.-methylmannose, the eluate was dialyzed against water, followed by lyophilization; the lyophilizate was then dissolved in phosphate buffer saline, and the solution was subjected to separation by isoelectric focusing, followed by concentration through ultrafiltration membranes to thereby purify uromodulin. Uromodulin, whose carbohydrate chain or moiety elicits numerous functions, has been considered to be a specific ligand for cytokines, such as IL-1, IL-2 and tumor necrosis factor (TNF) (Hession, C., et al., 1987, Science, 237: 1497. Sherblom, A. P., et al., 1989, J. Immunol., 143: 939, Brown, K. M., et al., 1986, Proc. Natl. Acad. Sci. U.S.A., 83: 9119, and Winkelstein, A., et al., 1990, Immunopharmacology, 20: 201). In recent years, however, what is purified from urine merely by the salting out method is referred to as "THG", and what is purified from pregnancy urine by the salting out method is called "uromodulin".
THG contains carbohydrates in quantity equivalent to about 30% of the molecular weight and possesses a carbohydrate chain structure being rich in mannose represented by GluNa.sub.2 and Man (5-7) wherein GluNa and Man denote N-acetylglucosamine and mannose, respectively. And it has been proven that the carbohydrate residues of Man 5 and Man 6 bind specifically to cytokines, inclusive of IL-1, IL-2 and tumor necrosis factor (TNF). These findings suggest the possibility that THG would be involved in the immunomodulatory mechanism, and at the same time, uromodulin showing the same amino acid sequence as THG was reported to exhibit immunosuppressive activity greater than 10 times more potent than THG. Also a report was recently published that THG of a sheep origin binds to sheep's IgG, while THG of a human origin likewise binds to human IgG (Rhodes, D. C. J. et al., 1993, Kidney Int., 44: 1014). Consequently, THG is considered to play a key role in novel immunomodulation or immune protection in the renal tubules, in addition to the immunoregulation mechanism mediated through cytokines.
In purifying THG and uromodulin, many researchers employ the above-mentioned salting out method. Nevertheless, the method incurs the risk of allowing not only the objective protein but also many other impurities to precipitate in entanglement with the protein, and this requires additional steps of removing such impurities to be provided in the subsequent purification procedure. In order to overcome such disadvantage, the objective protein must be precipitated under milder operating conditions. Furthermore, THG and uromodulin tend to gel readily in solution when such metal ions as Na.sup.+ and Ca.sup.++, coexist, and it is therefore far from being easy to dissolve the lyophilized THG in isotonic saline. For the reason of this, it is desirable to conduct purification of THG under physiological conditions from the beginning without incorporating the step of lyophilization in the purification process. Moreover, if THG and uromodulin individually are able to be assayed accurately and methods for quantitatively determining each of them are established accordingly, these would be considered to serve a useful purpose to clarify more efficiently their physiological and clinical significances.
The present inventors found that urine, whether it is ordinary urine or pregnancy urine, upon freezing and thawing, yields precipitates in large volume in which a great deal of THG is contained, and succeeded in allowing THG to precipitate in great quantities in one step of freezing and thawing without resorting to the salting out method. In contrast to the salting out method, moreover, the novel process does not cause rapid precipitation and was found to offer the advantage that impurities are less susceptible to entanglement in the precipitate. On the basis of simplification of the purification process by eliminating a number of steps required for the conventional counterparts, furthermore, the present inventors discovered that gel permeation by use of high performance liquid chromatography (hereinafter referred to briefly as "HPLC") can remove efficiently impurities (composed mainly of coloring matters), while the collected fraction containing the objective protein, after passing through, for example, a filter of about 0.2 .mu.m in pore size, can be stored at a temperature of 4.degree. C. without being freeze-dried. Referring to uromodulin, the inventors have established a process which enables the same to be purified from pregnancy urine by means of the benzoate precipitation method in addition to the above-described freezing-thawing precipitation method. It was also found that the purified THG binds to the human immunoglobulin classes of IgG, IgA and Ig M and all of the IgG subclasses of IgG1, IgG2, IgG3 and IgG4 as well as the F(ab').sub.2 fragment of IgG, whereas the purified uromodulin binds to the human immunoglobulin classes of IgG and IgA and the IgG subclasses of IgG1, IgG2 and IgG4, though it does not bind readily to IgM, the IgG subclass of IgG3 and the F(ab').sub.2 fragment of IgG.